This project is concerned with the mechanism of action of thymidylate synthetase (deoxyuridylate (dUMP) plus 5,10-methylenetetrahydrofolate (CH2FH4) yields thymidylate plus 7,8-dihydrofolate) as isolated from amethopterin-resistant Lactobacillus casei. The problems to be investigated include: (a) comparison of thiol-free enzyme, partially activated enzyme, and activated thymidylate synthetase in terms of specific activity, 5-fluoro-2'-deoxyuridylate (FdUMP)-CH2FH4-enzyme ternary complex formation, numbers of catalytic sulfhydryl groups, binary complex formation with dUMP and FdUMP, (b) separation and characterization of the protein precursors to the ternary complex forms employing covalent affinity chromatography or modification with highly charged sulfhydryl group reagents, (c) further characterization of the role of catalytic cysteines with reference to identification of a general base activating the catalytic sulfhydryl, determination of the status of the catalytic cysteine in binary complex formation, and investigation of the mechanism of inhibition produced by 1,1,1-trifluoro-3-bromopropane, and (d) studies pertaining to the role of arginyl residues in thymidylate synthetase.